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Published on: August 13, 2009
Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization
Abstract:
Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable.
Insights
This study evaluated fixatives for in situ hybridization in bulky tissues. Formalin fixation, commonly used for routine processing, is compatible with in situ hybridization, enabling retrospective studies.
Area of Science:
- Veterinary Pathology
- Molecular Biology
- Histology
Background:
- Traditional in situ hybridization (ISH) methods use small tissue samples and electron microscopy fixatives like periodate-lysine-paraformaldehyde (PLP).
- These fixatives have poor penetration and hardening capabilities, limiting their use with larger tissues such as whole brains.
- The compatibility of fixatives suitable for bulky tissues with ISH needed evaluation.
Purpose of the Study:
- To assess fixatives adequate for large tissues, like pig and cow brains, for their compatibility with in situ hybridization.
- To determine if routine tissue processing methods could be used for retrospective viral disease studies using ISH.
Main Methods:
- BALB/c mice were intracranially inoculated with pseudorabies virus (PRV).
- Various fixatives were tested on brain tissue sections for in situ hybridization using a 35S-labeled PRV DNA probe.
- Tissue section loss and cellular morphology were assessed.
Main Results:
- Five fixatives supported positive in situ hybridization results.
- Periodate-lysine-paraformaldehyde (PLP) and MOCA fixative (a modified Carnoy's fluid) provided excellent cellular morphology.
- Formalin fixation, despite causing some tissue loss, proved compatible with in situ hybridization.
Conclusions:
- Several fixatives are suitable for in situ hybridization in larger tissue samples.
- Formalin-fixed, paraffin-embedded tissues can be used for retrospective in situ hybridization studies of viral diseases.
- This expands the possibilities for diagnosing and studying viral infections in veterinary pathology.
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